Current Medical Mycology 2018, 4(2): 21-26
Antifungal and antibacterial activities of polyherbal toothpaste against oral pathogens, in vitro Batool Sadeghi-Nejad1*, Eskandar Moghimipour2, Sedigheh Yusef Naanaie3, Shahrzad Nezarat4 1
Abadan School of Medical Sciences, Abadan, Iran Medicinal Plant Research Center, Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 3 Agricultural and Natural Resources Center, Ahvaz, Iran 4 Student Research Committee, Abadan School of Medical Sciences, Abadan, Iran 2
Article Info Article type: Original article
Article History: Received: 23 April 2018 Revised: 20 July 2018 Accepted: 06 August 2018
* Corresponding author: Batool Sadeghi-Nejad Abadan School of Medical Sciences, Abadan, Iran. Email:
[email protected]
ABSTRACT Background and Purpose: Herbal toothpastes are more secure and efficacious and less poisonous due to containing natural chemicals as compared with the synthetic toothpastes. The present study aimed to formulate a polyherbal toothpaste using accessible medicinal plants in Iran and evaluate its efficiency in the protection of oral hygiene and prevention of dental caries. Materials and Methods: The developed toothpaste was made of the leaf extracts of Artemisia dracunculus, Satureja khuzestanica (Jamzad), and Myrtus communis (Linn), combined at four different dilutions, namely 1:4 (25%), 1:1 (50%), 3:4 (75%), and (100%), with sterile distilled water. The product was tested against five microorganisms, including Streptococcus mutans, Lactobaccilus caseie, S. sanguis, S. salivarius, and Candida albicans, using agar well diffusion method. Results: After 24 h of incubation, the maximum mean diameters of inhibition zone against L. caseie and C. albicans were obtained as 17-30 and 10-25 mm, respectively. Furthermore, the minimum mean diameter of inhibition zone against S. salivarious was estimated as 15-20 mm. Conclusion: The formulated toothpaste showed potent inhibitory activities against Gram-positive bacteria and C. albicans. Therefore, more studies are required to accurately investigate the efficacy of the formulated toothpaste. Keywords: Antibacterial, Antifungal, Oral pathogens, Polyherbal toothpaste, Yeast
How to cite this paper Sadeghi-Nejad B, Moghimipour E, Yusef Naanaie S, Nezarat S. Antifungal and antibacterial activities of polyherbal toothpaste against oral pathogens, in vitro. Curr Med Mycol. 2018; 4(2): 21-26. DOI: 10.18502/cmm.4.2.65
Introduction ne of the most common chronic oral infections in the world is dental caries [1]. Oral pathogenic microorganisms have been the cause of dental plaques, dental caries, as well as gingival and periodontal diseases [2]. Streptococcus mutans is one of the main opportunistic pathogens of dental caries, which is responsible for the formation of dental plaque and caries [3]. Other microorganisms associated with this oral condition include Escherichia coli, S. aureus [4], and Candida species. C. albicans is the most frequent yeast isolated from the oral cavities with poor oral hygiene [2]. The formulation ingredients of commercial toothpastes are mostly chemical substances, such as fluoride and whitening agents [2]. The literature contains evidence regarding the adverse effects of fluoride and bleaching agents (e.g., peroxide-based agents) used in the commercial toothpastes [5]. With this background in mind, the present study aimed to formulate a polyherbal toothpaste without any
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fluoride or whitening agents and evaluate its antimicrobial properties. The main ingredients of this toothpaste included the leaf extracts of Artemisia dracunculus L. (ADL), Satureja khuzestanica (Jamzad; SKJ), and Myrtus communis (Linn; MCL). The MCL or myrtle, belonging to the Myrtaceae (Lamiaceae) family, is an aromatic evergreen small tree with small foliage and numerous branches [6]. In ancient medicinal herbs, myrtle leaves and flowers were used for the treatment of respiratory problems, dysentery, urinary tract infections, and candidiasis as a mouthwash [7]. According to the literature, the essential oil of MCL has pharmacological activities, including antioxidant [8], antimicrobial [9, 10], and antifungal activities [11, 12]. There is evidence regarding the inhibitory activity of the essential oil of MCL against clinically isolated oral pathogenic microorganisms [13]. Artemisia is a small and continual aromatic shrub from the Asteraceae family [14], which is called "Tarkhon" in Iran. In traditional medicine, this plant is used for the
Copyright© 2018, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY) License (http://creativecommons.org/) which permits unrestricted use, distribution and reproduction in any medium, provided appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Antimicrobial properties of polyherbal toothpaste
remedy of stomach pains, fever, and diabetes and is known to have anti-inflammatory, anti-parasitic, antioxidant, and antimicrobial activities [15]. On the other hand, SKJ, belonging to the Lamiaceae family, is extensively grown in the northern Khuzestan and southern Lorestan provinces of Iran [16]. This plant has traditionally been used for relieving tooth pain, strengthening the gum, and healing the wound in the southern part of Iran [17]. Moreover, this herb has been applied for antimicrobial [18] and antifungal [19] purposes, as well as the treatment of infectious diseases [20-21]. With regard to the previous studies reporting the antimicrobial properties of polyherbal toothpaste [22, 23], the current study was conducted to formulate a new polyherbal toothpaste containing the aqueous herbal extracts available in Iran and evaluate its antimicrobial potency against oral pathogens.
Materials and Methods Preparation of extracts The ADL was purchased from the local market, and MCL and SKJ were prepared with the aids of the Agricultural and Natural Resources Center, Ahvaz, Iran. The collected plants were air dried in shade. About 10 g of each powdered air dried plant was added to100 ml sterile distilled water (1:1 W/V) in a glass beaker for maceration, and then incubated on a rotary shaker for 72 h [24]. In the next step, the filtration of suspension was accomplished using Whatman filter paper No.1. The filtrated aqueous extracts were evaporated and dried at the room temperature. The extracts were stored in air-tight containers at -20˚C until future use. Microorganisms and inoculum preparation The antimicrobial activity of four anaerobic bacteria (Gram-positive) isolated from clinical isolates and Candida species was assessed. A total of twelve anaerobic bacteria, including S. sanguis, S. salivarius, S. mutans, and L. casei, were prepared from the frozen stock cultures obtained from the Department of Medical Microbiology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. In addition, five C. albicans strains were isolated from patients with periodontitis and gingivitis referring to the Educational
Sadeghi-Nejad B et al.
Clinics of Dentistry School, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. These samples were subcultured, and then diluted in a sterile normal saline solution (0.9%) to obtain a concentration of 5×105 CFU/ml for fungal strains and a colony forming unit of 106 (CFU/ml) for bacterial strains, adjusted with the turbidity of 0.5 McFarland [25, 26]. Formulation of polyherbal toothpaste The ingredients of the fluoride-free polyherbal toothpaste was prepared according to the procedure adopted by Sekar and Zulhilmi Abdullah [22] with some modifications (Table 1). Screening polyherbal toothpaste for antimicrobial activity The formulation of the toothpaste was accomplished using three plants, namely ADL, MDL, and SKL, which were previously confirmed to have antimicrobial activities by in vitro assays. The dentifrice solution was prepared according to the previously reported procedures [2, 27]. The solution was tested against five microorganisms, including S. mutans, L. caseie, S. sanguis, S. salivarius, and C. albicans, by using agar well diffusion method following the previous studies [28, 29]. The agar plates inoculated with bacteria were kept in an anaerobic cabinet supplied with CO2 at 37°C for 24, 48, and 72 h. On the other hand, those agar plates inoculated with C. albicans were incubated at 30°C for 48 h [30]. Statistical analysis Statistical analysis was performed in SPSS software (version 20.0). The mean diameters of the inhibition zones were calculated. P-value less than 0.05 was considered statistically significant. Quality parameters of formulated polyherbal toothpaste The organoleptic investigation of polyherbal toothpaste, including color, taste, odor, and texture, were carried out by sensational and visual surveys according to the modified procedure of Sekar and Zulhilmi Abdullah [22].
Table 1. Ingredients of the formulated polyherbal toothpaste Components Amounts g/% A. dracunculus leaf extract 0.0625 S. khuzestanica leaf extract 0.0625 M. communis leaf extract 0.0625 Hydroxypropyl methyl cellulose 3 Sodium lauryl sulphate 5 Calcium carbonate 25 Glycerin 5 Methyl paraben 0.5 Propyl paraben 0.25 Sodium saccharine 0.3125 Peppermint oil 0.75 (2-3 drops) Demineralized water 60.5 Total 100 ml
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Property Active ingredient Active ingredient Active ingredient Gelling agent Surfactant Abrasive Anti-crusting agent Preservative Preservative Sweetener Flavoring agent Vehicle
Curr Med Mycol, 2018, 4(2): 21-26
Sadeghi-Nejad B et al.
Antimicrobial properties of polyherbal toothpaste
Results Table 2 summarized the inhibition zones produced by four polyherbal toothpaste dilutions of full strength, 3:4, 1:1, and 1:4 against S. mutans, S. salivarius, S. sanguis, L. casei, and C. albicans. The mean values of the microbial inhibition zones are shown in Table 2. The results demonstrated that L. casei showed the highest sensitivity against the dilutions of polyherbal toothpaste ranging from 5-30 mm in 24 h, followed by C. albicans (10-25 mm), S. mutans (5-25 mm), S. salivarius (10-25 mm), and S. sanguis (5-20 mm) at different dilutions of the toothpaste (figures 1 and 2; Table 2). All dilutions of polyherbal toothpaste were effective in inhibiting the
growth of the tested bacterium and fungus; however, they had no inhibitory effect on L. caseie, S. sanguis, and S. mutans at the dilution of 1:4. The polyherbal toothpaste showed a significant antimicrobial activity against all tested bacterium and yeast (P
